A-C) Free energy contour maps of DapA as a function of RMSD and ρ for three different temperatures, namely; 310 K, 360 K and 400 K, respectively. The color bar denotes the Gibbs free energy in kJ/mol. The inset within the maps show distribution of fraction of native contacts, Q. Q is defined by the total number of native contacts for each trajectory frame divided by the total number of contacts in the native structure. D) Thermal melting curve of DapA derived from CD (in black) and REMD simulations (in green), is depicted (see for details). The molar ellipticity values obtained at 222 nm were normalized between 0 to 1 as a function of temperature. E-F) displays RMSD distributions of ρ-RMSD, Q-RMSD SASA-RMSD maps for I1 and I2 configurations, respectively. The RMSD was calculated with respect to the native structure.

A) Representative snapshots showing disruption of TIM barrel topology with α and β-region shown in blue and red color, respectively. B) Time evolution of distance between α and β-core for all three 400 K simulation shown in three shades of orange. For comparison, native 300 K is also shown in black. C) Time occurrence of representative β-core residues. The brown color represents the existence of beta-sheet secondary structure based on the dihedral angles.

A) Probability of each α and β secondary structural element normalized to the native protein structure as a function of amino acid residues is calculated. Comparison of I1 and I2 with N (native) reveals multiple conformational changes of β-sheets and α-helices as marked above in blue and green respectively. The secondary structures were assigned with DSSP. Error bars denote the standard deviation calculated from three simulations. B) The top panel shows the time occurrence of α-helix (magenta) to β-sheet (green) transition observed in α4 helix in one of the representative trajectory. In addition, α4 hydrogen bonds (in blue) and dihedral angle of His118 belonging to α4 (below) as a function of time are displayed, to indicate rearrangement of local bonding patterns.

A) Emission spectra of free 1–8 ANS (final 10 μM) in buffer solution and bound to DapA incubated at 308 K and 333 K are plotted. The spectra was recorded after incubation with DapA (final 2 μM) subjected to thermal denaturation by gradual increase in temperature. B) “Exposed Hydrophobic Surface Contribution” (EHSC) as a function of major conformational transition events in I1 and I2 are plotted. Four primary events contribute to conformational transitions in DapA, namely, i) α-helix to random coil ii) β-sheet to random coil iii) random coil to β-sheet, and iv) α-helix to β-sheet. The contribution of each event is attributed to the surface exposed hydrophobic patches i.e., how much percentage of α-helix to random coil event is giving rise to the total hydrophobicity in intermediate structures. C-E) Representative snapshots of N, I1 and I2 showing a clear increase in exposed hydrophobic patches in intermediates with respect to native, where exposed non-polar atoms of the contributing residues are highlighted in red color.

Representative snapshot of the native (A) and the I2 structure (B), displaying IVL residues constituting a part of GroES-like binding motifs. In comparison to the native structure, these clusters are mostly solvent-exposed.

A-C) Distribution of C-α distances of three crucial non-native interactions in I2: Val191-Leu151, His53-Tyr107, and His118-Leu114, as shown in blue, red, and magenta, respectively. The native protein distance distribution is shown in gray, where these non-native contacts are largely absent. D-E) Representative snapshots of the native and I2 displaying the corresponding residues in colored stick representation.

A) Protein structures of DapA and TIM, highlighting their similar TIM barrel topology. B) Free energy contour map of TIM as a function of RMSD and ρ at 400 K as a control is depicted. The color bar denotes the Gibbs free energy in kJ/mol. The inset within the map shows distribution of fraction of native contacts, Q.

Network graphs of N, I1, and I2 are shown in panel A-C, respectively. The nodes represents the amino-acid residues, communication pathways are depicted in bold lines and two connected residues by thin lines. Residues are colored from dark to light violet according to their communication efficiency, calculated by the number of residues to which they are connected. High communication between interacting residues describe pathways of well-defined interactions and such chains of residues constitute the communication pathway through which signals are transmitted efficiently. D-F) Snapshots of native and intermediates, highlighting the stable cluster within each structure. The 2D and 3D graphs are drawn with GEPHI and CHIMERA. The communication pathways are calculated using the MONETA tool.